Description. Streptococcus pneumoniae (Spn, the pneumococcus), a gram-positive human pathogen, is a significant cause of morbidity and mortality worldwide. Pneumococcus causes more deaths than any other infectious disease, with children and the elderly at the highest risk. Epidemiological data suggest host-to-host transmission of Spn is the critical first step required for both the carrier- and the disease-state, suggesting that it is imperative to block host-to-host events. However, because of the inherent complexities in studying natural transmission and the absence of tractable animal models, Spn transmission is one of the least understood aspects of this pathogen's lifecycle. We developed a tractable infant mouse model that has allowed for the identification of host and bacterial factors that contribute towards the transmission process. More recently, we screened random pools of transposon mutants (Tn-seq) and identified novel Spn genes whose products are involved in host-to-host transmission. This list of Spn transmission factors included YesMN, a poorly defined two-component system (TCS). Generally, TCS mediate rapid transcriptional changes in response to external stimuli. Hence, our focus in this proposal is the TCS YesMN. We premise that within the upper respiratory (URT), pneumococcus undergoes transcriptional alterations facilitated by YesMN that sense the host environment and respond accordingly, allowing for Spn persistence and enable it to transit from one host to another. We identified the putative regulon of YesMN through an in vitro RNA-seq screen, which included genes involved in zinc (Zn) homeostasis. Thus, in Aim #1, we will follow up on these putative regulon members and determine their contribution to the transmission process. We will also test whether Zn, a highly regulated metal by the host, acts as a signal for YesMN. As pneumococcal pneumonia is considered a clinical complication of influenza A virus (IAV) infection, we further tested whether YesMN affects Spn fitness under the Spn-IAV coinfection setting. We observed that with a concurrent IAV infection, Spn modifies its transcriptome, with a significant contribution from YesMN, which provides a fitness advantage in the URT. By taking a separate approach in Aim #2, we will carry out a novel in vivo RNA-seq screen on samples obtained from the URT and determine YesMN regulated genes that are potentially involved in providing fitness to Spn under these dynamic coinfection conditions. Results from our current studies would, for the first time, provide an understanding of the Spn transcriptional dynamics occurring in the URT that promote host-to-host transmission and whether the identified factors could be potential targets to reduce pneumococcal disease burden.